title: Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.
abstract: Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.
authors.affiliation: National Center for Molecular Characterization of Genetically Modified Organisms, SJTU-Bor Luh Food Safety Center, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Huali
authors.initials: H
authors.lastname: Huang
authors.forename: Fang
authors.initials: F
authors.lastname: Cheng
authors.forename: Ruoan
authors.initials: R
authors.lastname: Wang
authors.forename: Dabing
authors.initials: D
authors.lastname: Zhang
authors.forename: Litao
authors.initials: L
authors.lastname: Yang
mesh_terms: D017931:DNA Primers / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics*; D014644:Genetic Variation; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013214:Starch Synthase / Q000235:genetics; D014908:Triticum / Q000235:genetics*
chemical_list: D017931:DNA Primers; D010940:Plant Proteins; C060756:waxy protein, plant; D013214:Starch Synthase